A HPLC fingerprint detection method for Qingyanlihou tablets

Abstract

This invention belongs to the field of traditional Chinese medicine quality testing technology, specifically relating to an HPLC fingerprint detection method for Qingyan Lihou tablets. The method includes using high-performance liquid chromatography (HPLC) to detect Qingyan Lihou tablets. The chromatographic conditions include: using a 1% (w / w) aqueous acetic acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution. The gradient elution procedure is as follows: This invention effectively separates various chromatographic peaks, resulting in numerous characteristic peaks with good peak shape and good separation. It allows for relatively accurate detection, exhibiting good detection stability, consistency, and controllability, and can comprehensively and reliably detect the quality of Qingyan Lihou tablets.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine quality testing technology, specifically relating to an HPLC fingerprint detection method for throat-clearing and throat-soothing tablets. Background Technology

[0002] The Qingyan Lihou tea bag is an experienced formula developed by Wei Xiangming, a renowned traditional Chinese medicine doctor at the Hunan Provincial Academy of Traditional Chinese Medicine. It consists of Rehmannia glutinosa, Ophiopogon japonicus, Fritillaria thunbergii, Platycodon grandiflorus, Adenophora stricta, Paeonia lactiflora, Paeonia suffruticosa, Mentha haplocalyx, Scrophularia ningpoensis, Glycyrrhiza uralensis, Terminalia chebula, and Chrysanthemum morifolium, and has the effects of nourishing Yin, clearing heat, moistening the lungs, and relieving cough. This formula obtained its pharmaceutical approval number in 1997 and has been used clinically ever since, showing unique efficacy in treating chronic pharyngitis. However, because it is a tea bag, it needs to be brewed and is inconvenient to carry, making long-term use unsuitable. Throat lozenges, as an oral preparation, are convenient to carry and take, act directly on the throat mucosa, quickly relieve symptoms, improve patients' quality of life, and reduce dependence on other medications. Therefore, changing the Qingyan Lihou tea bag to a throat lozenge form is of great significance for clinical application.

[0003] The preparation method of Qingyan Lihou Tablets is as follows: The extract is concentrated and dried to obtain a dry paste. The dry paste is combined with raw medicinal powder and excipients in a certain proportion, softened with 85% ethanol, and the granules are dried at 50-60℃, sieved, and 0.5% magnesium stearate is added as a lubricant before tableting. The extract is obtained by water extraction. The extracted Chinese herbal formula is: Rehmannia glutinosa 3.6g, Platycodon grandiflorus 0.9g, Paeonia lactiflora 2.7g, Paeonia suffruticosa 2.7g, Mentha haplocalyx 0.9g, Fritillaria thunbergii 1.35g, Scrophularia ningpoensis 2.7g, Glycyrrhiza uralensis 0.9g, Adenophora stricta 2.7g, Terminalia chebula 2.7g, Ophiopogon japonicus 2.7g, and Chrysanthemum morifolium 1.35g. Half the dose of Paeonia suffruticosa and the entire dose of Mentha haplocalyx are pulverized into a fine powder for later use as raw medicinal powder. The water extraction process is as follows: Half the dose of peony bark is added to 10 other Chinese medicinal herbs, including rehmannia root. The first decoction is prepared with 10 times the amount of water and decocted for 2.0 hours. The second decoction is prepared with 9 times the amount of water and decocted for 1.5 hours. The concentration conditions for the extract are: temperature 60–70℃, vacuum degree -0.06–-0.1 MPa. The drying conditions for the extract are: temperature 60–70℃, vacuum degree -0.06–-0.1 MPa. The excipient is soluble starch, used in the following ratio: dry extract powder: raw herb powder: soluble starch = 1:0.2892:0.64. The wetting agent is 85% ethanol (volume concentration), used at 30%–35%. Flavoring agents can be added, namely 1% steviol glycosides and 0.19% menthol.

[0004] With the increasingly widespread application of traditional Chinese medicine (TCM), current single-component control methods are no longer sufficient to meet the requirements of overall preparation quality control. Characteristic chromatograms can reflect the chemical components and distribution characteristics of TCM, as well as their distribution ratios, content characteristics, potential physiological activities, and quantitative efficacy characteristics. This aligns with the holistic, characteristic, multi-target, and non-linear characteristics that should be reflected in TCM quality control. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an HPLC fingerprint detection method for throat lozenges, thereby improving the accuracy of throat lozenge quality detection.

[0006] This invention provides an HPLC fingerprint detection method for throat-clearing and throat-soothing tablets, including the detection of the tablets using high-performance liquid chromatography. The chromatographic conditions include: using a 1% (w / w) aqueous acetic acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution. The gradient elution procedure is as follows:

[0007]

[0008] Preferably, the flow rate is 1.0 mL / min under the chromatographic conditions.

[0009] Preferably, the column temperature is 30℃.

[0010] Preferably, the detection wavelength is 254nm.

[0011] Preferably, the chromatographic column used is an Ultimate XB-C18.

[0012] Preferably, the test solution is prepared by taking throat lozenges, grinding them, adding methanol, sonicating, and filtering to obtain the test solution.

[0013] Preferably, the weight-volume ratio of the throat lozenge to methanol is 2g:25mL.

[0014] Preferably, the ultrasonic treatment power is 300W and the frequency is 40kHz.

[0015] Preferably, the method further includes high-performance liquid chromatography (HPLC) detection of the reference solution, wherein the reference standard is gallic acid, chlorogenic acid, paeoniflorin, glycyrrhizin, and paeonol.

[0016] The beneficial effects of this invention are as follows: Through experiments, this invention has found that when using high performance liquid chromatography to detect Qingyan Lihou tablets, using a 1% (w / w) aqueous solution of acetic acid as mobile phase A and acetonitrile as mobile phase B, and performing gradient elution as described in this application, can effectively separate the various chromatographic peaks, resulting in more characteristic peaks, better peak shapes, and better separation. This allows for more accurate detection, better detection stability, consistency, and controllability, and enables more comprehensive and reliable detection of the quality of Qingyan Lihou tablets. Attached Figure Description

[0017] Figure 1 This is a feature map of Embodiment 1 of the present invention.

[0018] Figure 2 This is the feature map of Comparative Example 1 of the present invention. Figure 3 This is the feature map of Comparative Example 2 of the present invention. Figure 4 This is the feature map of Comparative Example 3 of the present invention. Figure 5 This is the feature map of Comparative Example 4 of the present invention. Figure 6 This is the feature map of Comparative Example 5 of the present invention. Figure 7 This is the feature map of Comparative Example 6 of the present invention. Figure 8 This is the feature map of Comparative Example 7 of the present invention. Figure 9 This is the feature map of Comparative Example 8 of the present invention. Figure 10 This is the feature map of Comparative Example 9 of the present invention. Figure 11 This is the feature map of Comparative Example 10 of the present invention. Figure 12 This is the feature map of Comparative Example 11 of the present invention. Figure 13 This is a feature map of Comparative Example 12 of the present invention.

[0019] Figure 14 The characteristic spectrum of Example 2 at a wavelength of 254 nm is shown below. Figure 15 The characteristic spectrum at a wavelength of 327 nm is shown in Example 2. Figure 16 The characteristic spectrum of Example 2 at a wavelength of 210 nm is shown below. Figure 17 The characteristic spectrum of Example 2 at a wavelength of 230 nm is shown below. Figure 18 The characteristic spectrum of Example 2 is shown at a wavelength of 280 nm.

[0020] Figure 19 The image shows the characteristic spectrum of the extraction solvent in Example 3.

[0021] Figure 20 This is the characteristic spectrum of the test sample solution in Example 4. Figure 21 The characteristic chromatogram of the detection reference solution in Example 4 is shown below. Figure 22 This is a characteristic spectrum of the detection blank solution in Example 4.

[0022] Figure 23 This is the chromatogram of a double-negative sample lacking both white peony root and peony bark from Example 4. Figure 24 This is the chromatogram of the negative sample lacking white peony root from Example 4. Figure 25 This is the chromatogram of the negative sample lacking peony bark in Example 4. Figure 26 This is the chromatogram of the negative sample lacking chrysanthemum in Example 4. Figure 27 This is the chromatogram of the licorice-deficient negative sample from Example 4. Figure 28 This is the chromatogram of the negative sample lacking Scrophularia ningpoensis from Example 4. Figure 29 This is the chromatogram of the Rehmannia glutinosa-deficient negative sample from Example 4. Figure 30 This is the chromatogram of a negative sample of fruit lacking green leaves from Example 4. Figure 31 This is the chromatogram of a negative sample of *Adenophora stricta* from Example 4. Figure 32 This is the chromatogram of the peppermint-deficient negative sample from Example 4. Figure 33 The peak assignments for the characteristic spectrum of Example 4 are shown. Detailed Implementation

[0023] Instruments and reagents

[0024] (1) Instruments

[0025] Agilent Technologies 1260Infinity II high-performance liquid chromatograph with DAD detector; SB-5200D ultrasonic cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); Lingsheng multi-functional pulverizer (Yongkang Hongtaiyang Electromechanical Co., Ltd.); ES-502HA electronic balance (Changsha Xiangping Technology Development Co., Ltd.); SQP electronic analytical balance (Sartorius Scientific Instruments (Beijing) Co., Ltd.); XPE105 electronic analytical balance (METTLER).

[0026] (2) Drug testing

[0027] The preparation method of Qingyan Lihou tablets is as follows: the extract is concentrated and dried to obtain a dry paste. The dry paste is combined with raw medicinal powder and excipients in a certain proportion, and 85% ethanol is used to make a soft material. The granules are dried at 50-60℃, sieved, and 0.5% magnesium stearate is added as a lubricant. The tablets are then compressed.

[0028] The extraction was performed by water extraction. The extracted herbal formula consisted of: 3.6g Rehmannia glutinosa, 0.9g Platycodon grandiflorus, 2.7g Paeonia lactiflora, 2.7g Paeonia suffruticosa, 0.9g Mentha haplocalyx, 1.35g Fritillaria thunbergii, 2.7g Scrophularia ningpoensis, 0.9g Glycyrrhiza uralensis, 2.7g Adenophora stricta, 2.7g Terminalia chebula, 2.7g Ophiopogon japonicus, and 1.35g Chrysanthemum morifolium. Half the dose of Paeonia suffruticosa and the entire dose of Mentha haplocalyx were ground into a fine powder and set aside as raw herb powder. The water extraction process was as follows: Half the dose of Paeonia suffruticosa was decocted with the remaining 10 herbs, including Rehmannia glutinosa, for 2.0 hours with 10 times the amount of water, and then decocted for 1.5 hours with 9 times the amount of water. The concentration conditions for the extract were: temperature 60–70℃, vacuum degree -0.06–-0.1 MPa; the drying conditions for the extract were: temperature 60–70℃, vacuum degree -0.06–-0.1 MPa; the excipient was soluble starch, and its dosage ratio was: dry extract powder: crude drug powder: soluble starch = 1:0.2892:0.64; 85% ethanol (volume concentration) was used as a wetting agent, and its dosage was 30%–35%.

[0029] Acetonitrile and methanol were of chromatographic grade, water was purified water, and all other reagents were of analytical grade.

[0030] Reagents: Chromatographic methanol (Sigma-Aldrich, 99.9%), chromatographic acetonitrile (Sigma-Aldrich, 99.9%), purified water (Yibao purified water), analytical methanol (Sinopharm Chemical Reagent Co., Ltd.), analytical ethanol (Sinopharm Chemical Reagent Co., Ltd.), phosphoric acid (Shanghai Maclean Biochemical Technology Co., Ltd., 85-90%), glacial acetic acid (Shanghai Maclean Biochemical Technology Co., Ltd., 99.9%).

[0031] Reference standards: paeoniflorin (110736-202145, 99.9%, National Institutes for Food and Drug Control), paeonol (110708-201407, 99.9%, National Institutes for Food and Drug Control), chlorogenic acid (110753-201817, 96.8%, National Institutes for Food and Drug Control), glycyrrhizin (111610-202209, 95.2%, National Institutes for Food and Drug Control), gallic acid (110831-201906, 91.5%, National Institutes for Food and Drug Control).

[0032] Example 1

[0033] A high-performance liquid chromatography (HPLC) fingerprint detection method for Qingyan Lihou tablets is provided. The chromatographic conditions include: an Ultimate XB-C18 (4.6 × 250 mm, 5 μm) column; a flow rate of 1.0 mL / min; a column temperature of 30 °C; mobile phase A of 1% glacial acetic acid solution and mobile phase B of acetonitrile; gradient elution according to the specifications in the table below; washing and pre-equilibration for 35 min; and a detection wavelength of 254 nm. The gradient elution procedure is shown in Table 1.

[0034] Table 1. Characteristic map of Qingyan Lihou tablets and gradient elution procedure.

[0035]

[0036] The preparation method of the test solution is as follows: take an appropriate amount of throat lozenges, grind them into a fine powder, take about 2g, weigh it accurately, place it in a stoppered conical flask, add 25ml of methanol accurately, sonicate (power 300W, frequency 40kHz) for 30 minutes, take it out and let it cool, take the supernatant and filter it through a microporous filter to obtain the test solution.

[0037] Assay: Accurately pipette 10 μL of the test solution and inject it into the liquid chromatograph. Measure and record the chromatogram to obtain the result. Figure 1 As shown,

[0038] Experimental results are as follows Figure 1 As shown, the results indicate that the resolution of each chromatographic peak is good.

[0039] Comparative Example 1

[0040] The preparation method of the test solution is as follows: Take an appropriate amount of throat lozenges, grind them into powder and sieve them (80 mesh). Take about 2.0g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of pure methanol, stopper tightly, weigh it, sonicate (power 300W, frequency 40kHz) for 30 minutes, cool it, weigh it again, make up the lost weight with pure methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0041] High performance liquid chromatography (HPLC) was used to detect Qingyan Lihou tablets. The chromatographic conditions were as follows: Ultimate XB-C18 (4.6×250mm, 5μm) column, flow rate 1.0mL / min, column temperature 30℃, wavelength 256nm, injection volume 10μl, mobile phase A of 0.5% phosphoric acid solution and mobile phase B of acetonitrile, and gradient elution was performed according to the specifications in Table 2.

[0042] Table 2. Characteristic map of Qingyan Lihou tablets, gradient elution procedure 1

[0043]

[0044] Experimental results are as follows Figure 2 As shown, the results indicate that the chromatographic peaks are relatively dense and the resolution is insufficient during the 36-44 min time period.

[0045] Comparative Example 2

[0046] The difference between Comparative Example 2 and Comparative Example 1 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 2 is shown in Table 3.

[0047] Table 3. Throat-clearing and throat-soothing characteristic map, gradient elution procedure 2

[0048]

[0049] Experimental results are as follows Figure 3 As shown, the results indicate that the chromatographic peaks are relatively dense and the separation is insufficient during the 22-30 min time period.

[0050] Comparative Example 3

[0051] The difference between Comparative Example 3 and Comparative Example 1 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 3 is shown in Table 4.

[0052] Table 4. Throat-clearing and throat-soothing characteristic map, gradient elution procedure 3

[0053]

[0054] Experimental results are as follows Figure 4 As shown, the results indicate that the chromatographic peaks are relatively dense and the separation is insufficient during the 44-50 min time period.

[0055] Comparative Example 4

[0056] The preparation method of the test solution in Comparative Example 4 is the same as that in Example 1.

[0057] High performance liquid chromatography (HPLC) was used to detect Qingyan Lihou tablets. The chromatographic conditions were as follows: Ultimate XB-C18 (4.6×250mm, 5μm) column, flow rate 1.0mL / min, column temperature 30℃, wavelength 254nm, injection volume 10μl, mobile phase A of 1% glacial acetic acid solution and mobile phase B of acetonitrile, and gradient elution was performed according to the specifications in Table 5.

[0058] Table 5. Throat-clearing and throat-soothing characteristic map, gradient elution procedure four.

[0059]

[0060] Experimental results are as follows Figure 5 As shown, the results indicate that the chromatographic peaks are sparse.

[0061] Comparative Example 5

[0062] The difference between Comparative Example 5 and Comparative Example 4 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 5 is shown in Table 6.

[0063] Table 6. Throat-clearing and throat-soothing characteristic map, gradient elution procedure five.

[0064]

[0065] Experimental results are as follows Figure 6 As shown, the results indicate that some chromatographic peaks overlap.

[0066] Comparative Example 6

[0067] The difference between Comparative Example 6 and Comparative Example 4 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 6 is shown in Table 7.

[0068] Table 7. Throat-clearing and throat-soothing characteristic map, gradient elution procedure VII.

[0069]

[0070] Experimental results are as follows Figure 7 As shown, the results indicate that the chromatographic peaks are more concentrated in the 65-75 min time period, which is not as good as in program six.

[0071] Comparative Example 7

[0072] The difference between Comparative Example 7 and Comparative Example 4 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 7 is shown in Table 8.

[0073] Table 8. Throat-clearing and throat-soothing characteristic map, gradient elution procedure.

[0074]

[0075]

[0076] Experimental results are as follows Figure 8 As shown, the results indicate that the chromatographic peaks are generally correct.

[0077] Comparative Example 8

[0078] The preparation method of the test solution of Comparative Example 8 is the same as that of Comparative Example 7.

[0079] High performance liquid chromatography (HPLC) was used to detect Qingyan Lihou tablets. The chromatographic conditions were as follows: ShimNex CS C18 column (250 mm × 4.6 mm, 5 μm), flow rate 1.0 mL / min, column temperature 30 ℃, wavelength 254 nm, injection volume 10 μl, mobile phase A of 1% glacial acetic acid solution, and mobile phase B of acetonitrile, with gradient elution as specified in the table below.

[0080] Table 9. Throat-clearing and throat-soothing characteristic map, gradient elution procedure VI

[0081]

[0082] Experimental results are as follows Figure 9 As shown, the results indicate that the chromatographic peaks are generally good, but the peak shapes are not as good as those of the Ultimate XB-C18 column.

[0083] Comparative Example 9

[0084] The difference between Comparative Example 9 and Comparative Example 8 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 9 is shown in Table 10.

[0085] Table 10. Throat-clearing and throat-soothing characteristic map, gradient elution procedure 9

[0086]

[0087] Experimental results are as follows Figure 10 As shown, the results indicate that the chromatographic peaks are undesirable.

[0088] Comparative Example 10

[0089] The difference between Comparative Example 10 and Comparative Example 8 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 10 is shown in Table 11.

[0090] Table 11. Throat-clearing and throat-soothing characteristic map, gradient elution procedure.

[0091]

[0092] Experimental results are as follows Figure 11 As shown, the results indicate that the chromatographic peaks are undesirable.

[0093] Comparative Example 11

[0094] The difference between Comparative Example 11 and Comparative Example 8 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 11 is shown in Table 12.

[0095] Table 12 Throat-Clearing and Soothing Characteristic Atlas Gradient Washing Procedure Eleven

[0096]

[0097] Experimental results are as follows Figure 12 As shown, the results indicate that the chromatographic peaks are unsatisfactory, and further adjustments to the elution gradient should be attempted.

[0098] Comparative Example 12

[0099] The difference between Comparative Example 12 and Comparative Example 8 lies in the elution procedure; otherwise, they are the same. The elution procedure for Comparative Example 12 is shown in Table 13.

[0100] Table 13 Throat-clearing and throat-soothing characteristic map, gradient elution procedure twelve

[0101]

[0102] Experimental results are as follows Figure 13 As shown, the results indicate that the chromatographic peaks are undesirable.

[0103] Example 2

[0104] Wavelength selection

[0105] The difference between Example 2 and Example 1 is the wavelength; otherwise, they are the same. The wavelengths are 210nm, 230nm, 254nm, 280nm, and 327nm.

[0106] Chromatograms were compared at 210 nm, 230 nm, 254 nm, 280 nm, and 327 nm. The results are shown in [Figure number missing]. Figure 14-18 .

[0107] The results showed that the baseline was not stable at wavelengths of 210 nm and 230 nm, and the absorption signal of each peak above 280 nm decreased. At wavelengths of 254 nm and 327 nm, the peaks were relatively well separated, and more characteristic peaks were obtained with better overall peak shape. However, the standard sample was best at 254 nm, so the wavelength of 254 nm was selected.

[0108] Example 3

[0109] Investigation of the preparation method of the test solution

[0110] Extraction solvent

[0111] Take an appropriate amount of this product, 2g, in three portions, accurately weigh them, place them in a stoppered conical flask, and accurately add 25ml each of methanol, ethanol, and distilled water. Sonicate (300W power, 40kHz frequency) for 30 minutes, remove and cool, and filter the supernatant through a 0.22μm microporous filter to obtain the product.

[0112] Other conditions were the same as in Example 1, and the experimental results are shown below. Figure 19 .

[0113] During sample processing, ultrasonic treatment with water resulted in slow filtration. The number and area of ​​peaks in water-treated and ethanol-treated samples were significantly smaller than those in methanol-extracted samples.

[0114] Example 4

[0115] Methodological Validation

[0116] (1) Chromatographic peak identification

[0117] According to the HPLC fingerprint detection method in Example 1, the test solution, reference solution (gallic acid, chlorogenic acid, paeoniflorin, glycyrrhizin, paeonol), and solvent blank (methanol) of Example 1 were detected sequentially. The reference solution was prepared as follows: appropriate amounts of paeoniflorin, paeonol, chlorogenic acid, glycyrrhizin, and gallic acid reference standards were accurately weighed and added to methanol to prepare a solution containing 25.10 μg·mL of paeoniflorin. -1 Paeonol 28.51 μg·mL -1 26.54 μg·mL chlorogenic acid -1 Glycyrrhizin 20.30 μg / mL -1 Gallic acid 22.73 μg·mL -1 A mixed reference solution.

[0118] See results Figure 20-22 .

[0119] By comparing the peak times and the spectra of the reference sample and the test sample, it was determined that peak 1 in the figure is gallic acid, peak 3 is chlorogenic acid, peak 7 is paeoniflorin, peak 9 is glycyrrhizin, and peak 16 is paeonol, and the peak shape and resolution are relatively good.

[0120] (2) Peak attribution

[0121] According to the test sample preparation method in Example 1, 10 negative samples (in order of lack of white peony root and peony bark double negative sample, white peony root negative sample, peony bark negative sample, chrysanthemum negative sample, licorice negative sample, scrophularia negative sample, rehmannia negative sample, green fruit negative sample, north ginseng negative sample, and mint negative sample) and the test sample were processed.

[0122] Following the HPLC fingerprint detection method of Example 1, samples were injected and detected respectively, and the results are shown in [Figure 1]. Figure 23-33 .

[0123] The results showed that peaks 2, 5, 9, 10, 13, 15, and 16, a total of eight peaks, were tentatively selected as common peaks. Peaks 2, 5, and 9 were assigned to *Clematis chinensis*, peaks 10 and 13 to *Chrysanthemum morifolium*, peak 15 to *Mentha haplocalyx*, and peak 16 to *Paeonia suffruticosa*. No peaks were assigned to *Scrophularia ningpoensis* or *Rehmannia glutinosa*.

[0124] (3) Precision test

[0125] Prepare one sample solution according to the established analytical method, and inject it six times consecutively to examine the precision. Eight characteristic peaks were identified, with peak 16 (paeonol) as the reference peak. The relative retention times and relative peak areas of the other characteristic peaks were calculated, and the results are shown in Tables 14-15.

[0126] Table 14: Results of Relative Retention Time for Precision of Feature Maps

[0127]

[0128]

[0129] Table 15: Results of Relative Peak Area and Precision of Feature Maps

[0130]

[0131] The results showed that the relative retention time RSD of the eight common peaks did not exceed 1.0%, and the relative peak area RSD did not exceed 5.0%, indicating that the instrument precision was good.

[0132] (4) Repeatability test

[0133] Six test solutions were prepared according to the established analytical method and injected sequentially for analysis to examine repeatability. Twelve characteristic peaks were calibrated, with peak 16 (paeonol) as the reference peak. The relative retention times and relative peak areas of the other characteristic peaks were calculated, and the results are shown in Tables 16-17.

[0134] Table 16 Results of Relative Retention Time for Repeatability of Feature Maps

[0135]

[0136] Table 17 Results of Relative Peak Area for Repeatability of Feature Maps

[0137]

[0138] The results showed that the relative retention time RSD of the eight common peaks did not exceed 1.0%, and the relative peak area RSD did not exceed 5.0%, indicating that the method had good repeatability.

[0139] (5) Solution stability test

[0140] One test solution was prepared according to the established analytical method, and characteristic spectra were measured at 0h, 2h, 4h, 6h, 8h, 12h, and 24h to examine stability. Eight characteristic peaks were calibrated, and peak 16 (paeonol) was used as the reference peak. The relative retention times and relative peak areas of the other characteristic peaks were calculated, and the results are shown in Tables 18-19.

[0141] Table 18: Results of Relative Retention Time of Characteristic Spectrum Stability

[0142]

[0143] Table 19: Results of Relative Peak Area and Stability of Characteristic Spectra

[0144]

[0145] The results showed that the relative retention time RSD of the eight common peaks did not exceed 1.0%, and the relative peak area RSD did not exceed 5.0%, indicating that the solution had good stability within 24 hours.

[0146] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0147] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. An HPLC fingerprint detection method for throat-clearing and throat-soothing tablets, characterized in that, This includes the detection of Qingyan Lihou tablets using high-performance liquid chromatography (HPLC). The chromatographic conditions include: using a 1% (w / w) aqueous acetic acid solution as mobile phase A, acetonitrile as mobile phase B, and gradient elution. The gradient elution program is as follows: The detection wavelength was 254 nm, and the chromatographic conditions were as follows: the column was Ultimate XB-C18. During testing, the test solution is prepared by taking throat lozenges, grinding them, adding methanol, sonicating, and filtering to obtain the test solution. It also includes the detection of reference solutions by high performance liquid chromatography, wherein the reference standards are gallic acid, chlorogenic acid, paeoniflorin, glycyrrhizin and paeonol; The herbal formula of the throat-clearing tablets is as follows: Rehmannia glutinosa 3.6g, Platycodon grandiflorus 0.9g, Paeonia lactiflora 2.7g, Paeonia suffruticosa 2.7g, Mentha haplocalyx 0.9g, Fritillaria thunbergii 1.35g, Scrophularia ningpoensis 2.7g, Glycyrrhiza uralensis 0.9g, Adenophora stricta 2.7g, Terminalia chebula 2.7g, Ophiopogon japonicus 2.7g, and Chrysanthemum morifolium 1.35g.

2. The HPLC fingerprint detection method for throat-clearing and throat-soothing tablets as described in claim 1, characterized in that, The flow rate was 1.0 mL / min under the chromatographic conditions.

3. The HPLC fingerprint detection method for throat-clearing and throat-soothing tablets as described in claim 1, characterized in that, The column temperature is 30℃.

4. The HPLC fingerprint detection method for throat-clearing and throat-soothing tablets as described in claim 1, characterized in that, The weight-volume ratio of the throat lozenges to methanol is 2g:25mL.

5. The HPLC fingerprint detection method for throat-clearing and throat-soothing tablets as described in claim 1, characterized in that, The ultrasonic treatment power is 300W and the frequency is 40kHz.

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